Abortus Biovar Research Articles

Detection and characterization of Brucella species in rodents: A threat for the persistence of brucellosis in livestock farms

Brucellosis, caused by various Brucella species, poses a significant threat to global public health and livestock industries. This study aims to fill the knowledge gap concerning the presence of Brucella spp. in rodents on livestock farms in Iran. Both bacteriological and molecular surveys were conducted to assess the prevalence of Brucella spp. in these rodent populations. A total of 16 rodents were captured in four seropositive dairy cattle farms (n = 7) and two seropositive sheep farms (n = 9) and were then examined for the presence of the Brucella-infection. Five cow milk samples and 53 bovine lymph node samples from these farms were also tested for Brucella spp. Lymph node samples from dairy cattle farms contained 32 B. abortus biovar 3 isolates and one B. melitensis Rev1 vaccine isolate. The bacterial culture of rodents identified 12.5% of them (Mus musculus and Rattus norvegicus) harboring Brucella strains in dairy cattle farms. The rodents had B. abortus biovar 3 and B. melitensis biovar 1, suggesting a reservoir for these bacteria. A two-step molecular assay, utilizing the Omp28 sequences in tissue samples of rodents, demonstrated that 68.75% (n = 11) of the tested rodents yielded positive results. Bruce-ladder PCR and wboA typing on isolated bacteria revealed a close relationship to field strain of Brucella species. The study reveals that rodents on seropositive livestock farms in Iran harbor Brucella spp., indicating a potential reservoir for these bacteria. This highlights the importance of monitoring rodent populations through the molecular and bacterial methods to manage and control brucellosis in livestock.

Characterization of Brucella spp. circulating in industrial dairy cattle farms in Iran: a field study 2016 - 2023.

Bovine brucellosis, an infectious disease transmitted by Brucella melitensis and Brucella abortus, presents a significant zoonotic risk for agricultural economics and animal health. The primary objective of this study was to present a comprehensive understanding of the prevalence and features of Brucella strains within the industrial dairy farming sector in Iran. Rose Bengal plate test, standard agglutination test, and indirect enzyme linked immunosorbent assay tests were used to confirm all seropositive animals. A total number of 1,311 bovine samples from seropositive animals including were collected from 224 farms in 21 provinces of different regions of Iran and examined. The discovered Brucella isolates were phenotyped and molecularly characterized. The isolates were all B. abortus or B. melitensis. Bacteria analysis revealed that 70.53% of seropositive farms were tested positive for Brucella strains, predominantly B. melitensis biovar 1 (43.42%) and B. abortus biovar 3 (27.11%). Geographical distribution revealed that B. melitensis biovar 1 was the most common in dairy cow farms (16 provinces), followed by B. abortus biovar 3 (six provinces). Also, the prevalence of B. melitensis biovar 2, B. melitensis biovar 3, B. abortus biovar 1, B. abortus biovar 2 and RB51 vaccine were restricted to certain provinces. AMOS (abortus melitensis ovis suis)-polymerase chain reaction and Bruce-ladder PCR confirmed species identification. These results highlighted the complexity of bovine brucellosis in Iran and illustrated that B. melitensis was spread from small ruminants to cattle. This study provided important epidemiological insights for targeting future brucellosis control programs in the Iranian dairy farms.

Caracterização fenotípica e genotípica de isolados de Brucella abortus biovar 4 em bovinos no Brasil

ABSTRACT: The aim of the present study was to characterize (phenotypically and genotypically) two strains of Brucella abortus identified as belonging to biovar 4 isolated from cattle in Brazil. The strains were isolated from cervical bursitis from cattle in the states of Pará and Rio Grande do Sul, respectively. In the phenotypic identification, the isolates were positive in CO2 requirement, produced H2S, were resistant to basic fuchsin (20 µg / mL) and sensitive to thionin (20 µg / mL and 40 µg / mL) and presented M surface antigen, but A surface antigen is absent. The isolates were positive in the PCR for the bcsp31 gene (genus-specific) and in the AMOS-enhanced PCR, both isolates showed a band profile consistent with B. abortus biovar 1, 2 or 4. Moreover, both isolates also showed restriction patterns identical to the reference strain when tested by the omp2b PCR-RFLP. In genotyping using Multiple Locus Variable Number of Tandem Repeat (VNTR) Analysis - MLVA (MLVA16), the isolates showed differences in several loci (Bruce42, Bruce19, Bruce04, Bruce16 and Bruce30); by Multiple Locus Sequence Typing (MLST), they also exhibited differences in sequence type (ST), strain 16/02 ST1 (2-1-1-2-1-3-1-1-1) and strain 128/11 ST (22-1-1 -8-9-3-1-1-1). The extensive typing of B. abortus strains isolated from cattle in Brazil using different approaches confirmed the occurrence of rare B. abortus biovar 4 in the country.

The prevalence of camel brucellosis and associated risk factors

Camel brucellosis is a widespread and critical zoonotic infection caused by Brucella abortus and Brucella melitensis that negatively impact dairy and meat products. The current meta-analysis covers studies published over a course of 40 years, between 1 January 1980 and 1 April 2021, aiming to investigate the global prevalence of Brucella spp. in camels and related risk factors in different camel-keeping areas. Chi-squared test (I2 index) was applied to evaluate the heterogeneity of studies. A random-effect model (REM) estimated the pooled prevalence among subgroups. The literature search on camel brucellosis was performed among the available databases, including CABI, PubMed, Embase, Web of Science, Cochrane, Scielo, Scopus, and Science Direct. A total of 56 publications, comprising 205 data reports, were evaluated. Our results showed that the overall prevalence of camel brucellosis in the world was 9.23%. The lowest and highest prevalence rates of Brucella spp. infections among camels were reported in Oman (0.34%, 95%CI: 0.18–0.55) and Sudan (37.41%, 95%CI: 25.27–50.31), respectively. According to different risk factors, subgroup analysis showed that the location, sex, herd size, age, and mixed rearing with other animals could be considered the main risk factors impacting the occurrence of brucellosis in camels. The reported Brucella spp. in dromedary camel was Brucella melitensis (Biovar 1, 2, and 3), Brucella abortus (Biovar 3, 5, and 6), and Brucella suis. Our study represented remarkable differences between the prevalence of Brucella spp. in male (6.83% in 41 studies) and female (9.64% among 62 studies) camels. However, the present meta-epidemiological study would help improve control and surveillance approaches to prevent the spread of camel brucellosis among herds.

Occurrence and risk factors of brucellosis among domestic animals: an artificial neural network approach.

Brucellosis is a zoonotic infectious disease with a worldwide distribution. This cross-sectional study aimed to determine the occurrence of Brucella in milk and blood samples of ruminants and the importance factors associated with animal brucellosis in Eastern Iran. A total of 200 paired samples, including blood (100) and milk (100), were obtained from the goats, sheep, and cows in Eastern Iran. Serum agglutination (SAT) and 2-mercapto ethanol (2-ME) tests were performed on the sera. A multiplex-polymerase chain reaction (m-PCR) assay was performed to identify the following species of Brucella, including B. abortus biovar 1, 2, 4, 3b, 5, 6, and 9, B. abortus S19, B. melitensis, and B. melitensis Rev.1 vaccine strain. B. abortus RB51 vaccine strain was also investigated in a second PCR assay. Risk factors for infection with Brucella spp. including the effect of abortion, contact with the wild animals, herd type, age, and previous vaccination in predicting blood contamination with B. abortus biovar 1, 2, and 4 were modeled by use of the artificial neural network. A total of 23 samples were seropositive regarding SAT and 2-ME tests. In total, B. abortus was detected in 35% and 15% of blood and milk samples, respectively, by the m-PCR assay. One sample of each of milk and blood was detected to have B. melitensis. Some samples were simultaneously contaminated with two Brucella species or two biovars of B. abortus. B. abortus S19 and B. melitensis Rev.1 vaccine strains were also detected in milk and blood samples. The sensitivity and specificity of the ANN model were 81% and 62%, respectively. In conclusion, B. abortus had higher frequency than B. melitensis in blood and milk samples. ANN determined herd type, previous vaccination, and age of the animal as the largest predictors of blood contamination with B. abortus.

Genetic characteristics of an amikacin-resistant Brucella abortus strain first isolated from Marmota himalayana

Brucella spp. are facultative intracellular pathogens that can persistently colonize animal host cells and cause zoonotic brucellosis. Brucellosis affects public health and safety and even affects economic development. Our lab found that a Brucella strain isolated from Marmota himalayana exhibited amikacin resistance. To annotate and analyze the potential resistance genes in this strain, we utilized sequencing platforms in this study and cloned potential resistance genes. The findings showed that the isolated strain belonged to B. abortus biovar 1 and was similar to B. abortus 2308. The isolate had amikacin resistance genes encoding aminoglycoside 3′-phosphotransferase. Based on the results of genome analysis, the isolated strain may have obtained amikacin resistance genes from Salmonella spp. through Tn3 family transposons. Notably, this study establishes a foundation for further research on the resistance mechanism of Brucella spp. and provides data that may be useful for the prevention and control of drug-resistant Brucella strains.

In Vitro Evaluation of Immunogenicity of Recombinant OMP25 Protein Obtained from Endemic Brucella abortus Biovar 3 as Vaccine Candidate Molecule Against Animal Brucellosis.

Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. It was determined that doses of the recombinant Omp25 protein greater than 0.1 μg/mL are toxic to RAW cells. Doses of 1 μg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 μg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.

Isolation of Brucella abortus biovar 1 from human lumbar disc bulging: a case report of brucellar discitis

BackgroundBrucellosis is an endemic zoonotic disease with rising health and economic concerns in many areas worldwide. Musculoskeletal pains are among the main complications of human brucellosis, which are often difficult to diagnose due to the variability of clinical symptoms. Brucellar discitis is a very disabling problem in some chronic forms of the disease which may lead to serious vertebral and neurological consequences.Case presentationIn this case report, we reported the isolation of Brucella abortus from lumbar disc bulging in a woman who had rheumatoid arthritis and diabetes mellitus as underlying conditions. The patient had several negative brucellosis serological tests and dorsolumbar pains with urinary incontinence over a 2-month period. The diagnosis was confirmed by magnetic resonance imaging (MRI) examination of lumbar spine as well as disc culture. MRI examination was performed without intravenous contrast and revealed the presence of disc bulging, left foraminal narrowing at L5-S1, left foraminal narrowing, anterolisthesis grade II at L4-L5. The diagnosis was also confirmed by isolation of B. abortus biovar 1 from bulging disc culture. The isolate was characterized by AMOS PCR, Bruce-ladder PCR and biotyping, resulting in the identification of B. abortus from L4-L5 and L5-S1 disc bulging regions. The patient was treated with two drugs i.e. doxycycline and rifampin for 3 months. In the follow-up, in addition to improving the patient’s general condition, low-back pain was also significantly reduced.ConclusionsMRI, serology, cultural and molecular test along with patient history are important to make a rapid diagnosis of brucellosis’ discitis, thereby decreasing the delay for the brucellosis treatment. The present report suggests that the infection by Brucella spp. should be fundamentally considered among the causative agents of back pain especially in the endemic areas of Brucella infections.

Whole-Genome Sequencing for Tracing the Genetic Diversity of Brucella abortus and Brucella melitensis Isolated from Livestock in Egypt.

Brucellosis is a highly contagious zoonosis that occurs worldwide. Whole-genome sequencing (WGS) has become a widely accepted molecular typing method for outbreak tracing and genomic epidemiology of brucellosis. Twenty-nine Brucella spp. (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were isolated from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats originating from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR. Illumina MiSeq® was used to sequence the 29 Brucella isolates. Using MLST typing, ST11 and ST1 were identified among B. melitensis and B. abortus, respectively. Brucella abortus and B. melitensis isolates were divided into two main clusters (clusters 1 and 2) containing two and nine distinct genotypes by core-genome SNP analysis, respectively. The genotypes were irregularly distributed over time and space in the study area. Both Egyptian B. abortus and B. melitensis isolates proved to be genomically unique upon comparison with publicly available sequencing from strains of neighboring Mediterranean, African, and Asian countries. The antimicrobial resistance mechanism caused by mutations in rpoB, gyrA, and gyrB genes associated with rifampicin and ciprofloxacin resistance were identified. To the best of our knowledge, this is the first study investigating the epidemiology of Brucella isolates from livestock belonging to different localities in Egypt based on whole genome analysis.

Identification, geographic distribution and risk factors of Brucella abortus and Brucella melitensis infection in cattle in Algeria

Brucellosis is an infectious disease of several terrestrial and marine animals and humans caused by bacteria of the genus Brucella. This study aimed to identify Brucella species and biovars circulating in cattle and to analyze their geographic distribution across Algeria. Two hundred ninety eight milk and lymph node samples from 161 seropositive cattle of different local and foreign breeds were collected from 97 dairy farms in 56 towns of 13 wilayas (states/ provinces) of the central, eastern, western and southern regions. The samples were cultured on selective media and the obtained isolates were identified using bacteriological and molecular tests. Eighty-five Brucella isolates (72 B. abortus and 13 B. melitensis) were recovered from 63 animals in 37 dairy farms. In total, 71 (83.5 %) B. abortus bv 3, 11 (12.9 %) B. melitensis bv 2, 2 (2.4 %) B. melitensis bv 3 and 1 (1.2 %) unidentified B. abortus biovar were detected. The identification of B. abortus biovar 3 and B. melitensis biovar 2 is a new finding for Algeria and the Maghreb, respectively. B. abortus (84.7 %) was the main etiological agent of brucellosis. B. abortus showed a scattered distribution across Algeria. The fact that 60 % of the seropositive cattle showed no clinical signs, but 36 % were culture positive is an alarming observation. These data will rise awareness for the current epidemiological situation of bovine brucellosis in Algeria. To the best of our knowledge, this is the first representative countrywide bacteriological investigation of Brucella species and biovars in cattle across Algeria, which is a developing country where resources might be limited and the working conditions might not be very friendly.

Development of a Genus-Specific Brucella Real-Time PCR Assay Targeting the 16S-23S rDNA Internal Transcribed Spacer from Different Specimen Types.

The aim of this study was to develop a 16S-23S ribosomal deoxyribonucleic acid internal transcribed spacer (ITS) quantitative polymerase chain reaction (qPCR) assay for the early diagnosis and rapid screening of brucellosis. Blood, milk, and tissue samples were spiked with B. abortus biovar 1 (B01988-18 strain) to determine the analytical sensitivity and specificity of the assay. The 95% limit of detection of the ITS qPCR assay was highest in tissue, followed by blood, then milk, i.e., 0.48, 4.43, and 15.18 bacteria/PCR reaction, respectively. The diagnostic performance of the assay was compared to the Brucella cell surface protein (BCSP) 31 qPCR assay and bacterial culture. Out of 56 aborted foetal tissue samples from bovine, ovine, and caprine, 33% (19/56) were positive for Brucella spp. The sensitivity and specificity of the ITS qPCR assay was 87% and 95% respectively, compared to 92% and 89% for the BCSP31 qPCR assay and 47% and 55% for bacterial culture, respectively. The assay was efficient, sensitive, and specific, making it a valuable tool in the early detection of the Brucella pathogen.

Role of Brucella abortus Biovar 3 in the Outbreak of Abortion in a Dairy Cattle Herd Immunized with Brucella abortus Iriba Vaccine.

Bovine brucellosis is a widespread zoonosis caused by Brucella abortus. The disease is prevalent nationwide in Iran and is on an increasing trend among humans and livestock. The eradication of brucellosis is challenging and requires control policies at both national and regional levels. Regarding this, the aim of the current study was to evaluate if Brucella is implicated in an abortion outbreak that occurred in a dairy cattle herd, in Shahre Rey, Tehran province, Iran, after vaccination with B. abortus Iriba vaccine. The research context was a dairy cattle farm with 2,000 animals located in Shahre Rey. This farm was Brucella-free based on the results of two serological tests performed one month before vaccination. After the incidence of the first case of abortion following vaccination, serodiagnosis revealed a seropositive reaction in 30 non-pregnant cows and 19 pregnant cows that aborted later. Bacteriology and molecular typing facilitated the identification of 16 isolates of B. abortus biovar 3 from the aborted animals. None of the isolates were confirmed as B. abortus Iribavaccine strain. The results confirmed that B. abortus biovar 3 was the most prevalent biovar in the cattle of Iran. The source and time of infection in the current study were not detected most likely due to the low biosecurity level in the farm (e.g., uncontrolled introduction of the agents via humans, infected animals, semen, and vectors). In endemic countries, the serodiagnosis of brucellosis alone is not sufficient and has to be accompanied by isolation and molecular diagnosis. In addition, it is important to evaluate the presence of B. abortus in bovine semen and vectors.

Molecular epidemiology of Brucella abortus strains from cattle in Inner Mongolia, China

Although the prevalence of brucellosis in Inner Mongolia Autonomous Region currently remains high, data available on the epidemiological of circulating Brucella abortus strains were limited. A total of 75 isolates obtained from cattle, sheep, and humans were analysed using both the classical method and multiple locus variable-number tandem repeat analysis (MLVA). There are at least three B. abortus biovars (1, 3 and 6) in this region, and B. abortus biovar 3 is the predominant one. Ten known MLVA-11 genotypes were identified, of which five genotypes (72, 75, 78, 82 and 210) were shared among strains from this study and others previously collected in two to seven different nations, suggesting that this population has multiple geographic origins. An MLVA-16 assay sorted the 75 B. abortus strains into two groups (I and II), 5 clusters (A–E) and 44 genotypes (GT1-44), with 26 unique genotypes represented by single isolates, indicating that these B. abortus brucellosis cases were not directly epidemiologically related. The remaining 18 shared genotypes (among a total of 47 isolates) were represented by two to eight isolates, suggesting that there were epidemiologically related pathogens from each shared genotype among the cases. Importantly, the cluster B1 branch including 22 cluster isolates with identical or similar genotypes confirmed the occurrence of a concentrated outbreak epidemic in the eastern region during 1988–1995. This work will contribute to better understanding of B. abortus brucellosis epidemiology in Inner Mongolia.

First identification of a Brucella abortus biovar 4 strain from yak in Tibet, China

Brucellosis is one of the major zoonotic diseases in the world. In China, understanding on its causative agent Brucella is still limited. Recently, we isolated a Brucella strain XZ19-1 from yak in Lhasa, Tibet. Phenotypical characterization proved that it belongs to B. abortus biovar 4, a biotype that has never been reported in China. MLVA-16 genotyping revealed a novel profile (4-5-3-12-2-2-3-3-8-32-8-5-4-3-3-3) in this strain, while MLST sequence typing demonstrated that it belongs to ST 71. Furthermore, the whole genome of XZ19-1 strain was sequenced. Subsequent phylogenetic analysis demonstrated that XZ19-1was genetically more closely related to B. abortus strains originated from European countries rather than to those collected from China previously. Isolation and identification of XZ19-1 strain may thus indicate a unique Brucella lineage existing in Qing-Tibet plateau. These findings will help to improve the diagnosis and epidemiological studies of brucellosis in animals and human in this part of China.

Investigating selective media for optimal isolation of Brucella spp. in South Africa.

Bovine brucellosis in South Africa is caused mainly by Brucella abortus biovar (bv.) 1 and less frequently by B. abortus bv. 2. Bacterial isolation is regarded as the gold standard for diagnosis of Brucella species; however, it is not very sensitive. The aim of this study was to determine the selective medium with optimum antibiotic composition that will allow the growth of Brucella species (spp.) while inhibiting moulds, yeast and most, if not all, Gram-negative contaminants in South Africa. In the controlled experiment, modified Agrifood Research and Technology Center of Aragon (CITA) medium (mCITA) seemed to be the optimum selective medium for isolation of Brucella spp. as compared with Farrell’s medium (FM) and modified Thayer Martin (mTM), while FM inhibited the growth of most fungal and bacterial contaminants. Mean comparison between the three media used to culture B. abortus resulted in lower mean difference ranging from 0 to 2.33. In case of Brucella ovis, high mean difference was obtained when comparing FM with mCITA (10.33) and mTM (12). However, the mean differences of 0.67 and 1.67 were obtained when comparing mCITA and mTM media used to, respectively, culture pasteurised and raw milk spiked with B. ovis. Further optimisation at the Agricultural Research Council – Onderstepoort Veterinary Research Institute resulted in a comparable performance between FM and mCITA; however, mCITA allowed optimal growth of the fastidious B. ovis, which is generally inhibited on FM. Generally, mCITA seemed to be the optimum selective medium for isolation of Brucella spp., while FM inhibits the growth of most fungal and bacterial contaminants. Thus, veterinary laboratories can use mCITA and/or FM but should take into consideration the detection of factious Brucella isolated in the country or region.

COMPARISON OF HUMORAL IMMUNE RESPONSES BETWEEN HEAT-INACTIVATED BRUCELLA ABORTUS BIOVAR 3 AND STRAIN RB51 VACCINES IN INDIGENOUS CATTLE OF BANGLADESH

Background: Live attenuated Brucella abortus strains 19 and RB51 vaccines have been used as a key method for the control and eradication of brucellosis in cattle worldwide for decades. Due to certain limitations of these live vaccines, research has been undertaken for the development of an ideal more effective and safer vaccine for animals and human brucellosis. Objective: The main objective of this study was to compare the humoral immune responses (HIR) between the heat-inactivated Brucella abortus biovar 3 and attenuated live RB51 vaccines in native cattle of Bangladesh. Materials and Methods: The methods of isolation, identification, preparation of inoculum dose (10 × 1010 cfu/5 ml) and heat inactivation of B. abortus biovar 3 was followed as described earlier. Each of the three B. abortus sero-negative native cows was inoculated with heat-inactivated B. abortus vaccine @ 5.0 ml (10 × 1010 cfu /5 ml)/ cow SC single injection. Similarly, each of five native calves of 6 to 9 months old was inoculated with live attenuated RB51 vaccine (CZ Veterinaria, SA, Spain) @ 2.0 ml (10-34×109) SC as single dose. The sera of cows were collected at 0, 7, 14, 21, 28, 40, 60 and 90 days post vaccination, whereas the sera of the calves were collected at 0, 7, 14, 21, 28, 60, 90, 120, 150 and 180 days post-vaccination. All the collected sera of both the groups were tested to evaluate antibody titer by RBT followed by ELISA with commercial tests kits. Results: The HIR of the cows inoculated with heat-inactivated vaccine showed antibody (Ab) titer started to rise significantly (p < 0.05) from the 14 days (OD 0.2116 ± 0.0397, Ab titer 1:120) and reached a peak level at 28 days (OD 0.319 ± 0.172, Ab titer 1:800) and then started to decline significantly (p < 0.05) from 40 days (OD 0.234 ± 0.0415, Ab titer 1:35) to 60 days (OD 0.094 ± 0.0075, Ab titer 0). The mean Ab titer in calves inoculated with RB51 vaccine showed that Ab titer started to appears insignificantly (p ˃ 0.05) from day 7 (OD 0.094 ± 0.01603) and reached peak level at day 60 days (OD 0.592 ± 0.398), changes are very significant from day 0 (p < 0.05), after 60 days Ab level start to decrease and reach at lowest level at day 150 (OD 0.112 ± 0.0188), Ab level found similar to day 0 (OD 0.0826 ± 0.00517) at 180 days (OD 0.0822 ± 0.00249). Conclusions: The S19 and RB51 are the approved B. abortus vaccine strains have been widely and successfully used with some limitations to prevent bovine brucellosis worldwide. In addition to live attenuated and inactivated vaccines, recombinant genes, proteins, vectors, DNA and recombinant mutant vaccines have also been evaluated for the prevention of brucellosis but further research would be required to develop an ideal vaccine for both the humans and animals.

Identification, Genotyping and Antimicrobial Susceptibility Testing of Brucella spp. Isolated from Livestock in Egypt.

Brucellosis is a highly contagious zoonosis worldwide with economic and public health impacts. The aim of the present study was to identify Brucella (B.) spp. isolated from animal populations located in different districts of Egypt and to determine their antimicrobial resistance. In total, 34-suspected Brucella isolates were recovered from lymph nodes, milk, and fetal abomasal contents of infected cattle, buffaloes, sheep, and goats from nine districts in Egypt. The isolates were identified by microbiological methods and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Differentiation and genotyping were confirmed using multiplex PCR for B. abortus, Brucella melitensis, Brucella ovis, and Brucella suis (AMOS) and Bruce-ladder PCR. Antimicrobial susceptibility testing against clinically used antimicrobial agents (chloramphenicol, ciprofloxacin, erythromycin, gentamicin, imipenem, rifampicin, streptomycin, and tetracycline) was performed using E-Test. The antimicrobial resistance-associated genes and mutations in Brucella isolates were confirmed using molecular tools. In total, 29 Brucella isolates (eight B. abortus biovar 1 and 21 B. melitensis biovar 3) were identified and typed. The resistance of B. melitensis to ciprofloxacin, erythromycin, imipenem, rifampicin, and streptomycin were 76.2%, 19.0%, 76.2%, 66.7%, and 4.8%, respectively. Whereas, 25.0%, 87.5%, 25.0%, and 37.5% of B. abortus were resistant to ciprofloxacin, erythromycin, imipenem, and rifampicin, respectively. Mutations in the rpoB gene associated with rifampicin resistance were identified in all phenotypically resistant isolates. Mutations in gyrA and gyrB genes associated with ciprofloxacin resistance were identified in four phenotypically resistant isolates of B. melitensis. This is the first study highlighting the antimicrobial resistance in Brucella isolated from different animal species in Egypt. Mutations detected in genes associated with antimicrobial resistance unravel the molecular mechanisms of resistance in Brucella isolates from Egypt. The mutations in the rpoB gene in phenotypically resistant B. abortus isolates in this study were reported for the first time in Egypt.

Multilocus variable number tandem repeat analysis (MLVA)-typing of Brucella abortus isolates of India reveals limited genetic diversity.

Multilocus variable number tandem repeat analysis (MLVA) technique has wide applications in studying phylogenies and short-term epidemiology of pathogens. The technique has been extensively used worldwide in molecular epidemiology of Brucella genus. Only one study on this aspect is reported from India despite its economic and public health significance in country. The present study isolated B. abortus from domesticated bovines of Jammu region of Jammu and Kashmir state, India, and applied MLVA for 16 loci (MLVA-16). MLVA results were compared with the results of a previous study and with MLVA data of Indian isolates present in http://microbesgenotyping.i2bc.paris-saclay.fr/database. In the study, 136 samples from bovines (cattle and buffaloes) of 47 farms of Jammu region were processed for isolation. Eleven isolates of B. abortus biovar 1 from 6 farms were obtained. In MLVA-16 analysis, although the isolates were classified in a single cluster, 5 genotypes were obtained with a specific genotype being prevalent on each farm. The study identifies that MLVA-16 is capable to differentiate B. abortus strains in an area having high genetic similarity among isolates. On comparing the results with previous study and database, the isolates were found to have high genetic similarity indicating that the genetic diversity of B. abortus in India is very limited. It probably indicates that India is contaminated recently with B. abortus. To test this hypothesis, analysis of whole genome sequencing data of diverse collection of Indian B. abortus strains is required.

Serological, Isolation and Molecular Studies on Brucellosis in an Organized Farm, Jammu and Kashmir, India

The present study was conducted in an organized farm of Jammu region, Jammu and Kashmir state having history of late-term abortions in cattle and buffaloes to elucidate the role of brucellosis in causing abortions on the farm. The farm had 46 animals (23 adults, 15 heifers and 9 calves). The farm was positive in herd test (milk ring test). Serological tests viz., Rose Bengal plate test (RBPT) and serum agglutination test (SAT) were conducted on 30 serum samples and 11 samples were found positive in both tests. Five vaginal swabs from abortion cases were processed for isolation and 3 isolates of Brucella were obtained which on further characterization by biochemical tests, genus-specific PCR and species-specific PCR were identified as B. abortus biovar 1. As brucellosis is an anthropozoonoses, the high prevalence of brucellosis in livestock of farm demands the surveillance of disease in humans working on the farm and necessitates the control of disease on the farm.

A novel approach for detection of brucella using a real-time recombinase polymerase amplification assay

Brucella, the etiological agent of brucellosis, is an important zoonosis pathogen worldwide. Brucella infects humans and various domestic and wild animals, and represents a great threat to public health and animal husbandry. In the present study, we developed a real-time recombinase polymerase amplification (RPA) assay for the detection of Brucella. The assay targeted the bcsp31 gene of Brucella, and an RPA exo probe and a pair of primers were selected for assay validation. RPA sensitivity and specificity were evaluated using plasmid standards, Brucella representative strains, and non-Brucella strains. The RPA assay achieved a detection limit of 17 molecules in 95% of cases based on probit analysis, and could successfully distinguish 18 representative Brucella strains (B. abortus biovars 1, 2, 3, 4, 5, 6, 7 and 9, B. melitensis biovars 1, 2 and 3, B. suis biovars 1, 2, 3 and 4, B. canis, B. neotomae and B. ovis), and four Brucella vaccine strains (A19, S19, S2 and M5). A total of 52 Brucella field strains were detected by real-time PCR and RPA in parallel, and compared with real-time PCR, the sensitivity of the RPA assay was 94% (49/52). Thus, this RPA assay may be a rapid, sensitive, and specific tool for the prevention and control of Brucellosis.